Thrombosis is one of the leading causes of death world-wide. Cardiovascular events such as acute coronary syndromes as well as ischaemic cerebral infarctions are characterised by rupture or erosion of vulnerable atherosclerotic plaques and subsequent thrombosis. Thrombus formation impedes the flow of blood to vital organs and tissues, restricting oxygen supply and resulting ultimately in cell necrosis. It can be especially life threatening when this occurs in the lower body, heart, lungs or brain resulting respectively in deep vein thrombosis, acute myocardial infarction, pulmonary embolism or acute ischaemic stroke.
Various risk factors associated with atherosclerosis include hypercholesterolaemia, nitric oxide formation, smoking, as well as genetic factors. Thus certain individuals are at a higher risk of developing cardiac or vascular diseases than others.
Two pathways or coagulation cascades lead to the formation of a clot, known as the intrinsic and extrinsic pathways. These two pathways are initiated by distinct mechanisms but converge along a common pathway. Clot formation in response to an abnormal vessel wall in the absence of tissue injury is the result of the intrinsic pathway and clot formation in response to tissue injury is the result of the extrinsic pathway. The coagulation cascades are very complex and involve a number of different proteins known as clotting factors.
People who suffer from cardiac or vascular diseases and patients that have undergone surgical procedures are at risk of developing blood clots that may result in life-threatening clinical conditions. Such people are often treated with blood-thinning or anticoagulant drugs such as warfarin or aspirin. However, the amount of anticoagulant in the bloodstream must be maintained at the proper level: Too little may result in unwanted clotting whilst too much can result in haemorrhaging with life threatening consequences. As a result routine coagulation screening tests have been developed in order to evaluate the coagulation state of blood or plasma.
Clotting is a way by which the body closes off injured blood vessel walls following vascular injury. A blood clot consists of a plug of platelets enmeshed in a network of insoluble fibrin particles. The substance used in the blood to form a clot is fibrinogen, a protein synthesised by the liver which is cleaved by the enzyme thrombin to form fibrin peptides during normal coagulation. Thrombin also activates fibrin stabilising factor (Factor XIII) which subsequently cross-links the fibrin into a complex lattice. During clotting, fibrin strands start to form within the blood causing it to thicken. In time the thickened blood develop into a clot. Whilst formation of the clot is essential, the persistence of such clots is dangerous to the body. Thus, in order to minimise damage to the body after the clotting process has served its purpose, healthy cells surrounding the clot release plasmin to digest fibrin, therefore dissolving the clot.
A useful measure of coagulation is the so-called prothrombin time (PT) test and is routinely performed on patients who are on warfarin therapy following a cardiovascular event. The PT test measures the tissue factor- induced coagulation time of blood or plasma. This can provide an assessment of the extrinsic coagulation pathway and is sensitive to factors I, II, V, VII and X. The test is performed by adding a clotting agent such as thromboplastin and Ca2+ to a patient sample and then measuring the time for clot formation. Portable coagulation monitors such as the CoaguChek Plus™ coagulation meter have been developed which measure prothrombin time using non-anticoagulated capillary whole blood from a fingerstick or lancing device. Such monitors have been shown to be a valuable tool for patients on long-term oral anti-coagulation therapy.
However, the traditional expression of PT test results is inadequate for international comparison because the values depend upon the nature of the thromboplastin used. This has lead to the adoption of the Internationalised Normalised Ratio or INR as a way of expressing prothrombin time. INR is defined byINR=(observed PT ratio)exp ISI
where ISI is the International Sensitivity Index and PT ratio=Patient's PT/Mean Normal PT.
The ISI is derived from the calibration line of the value of PT for a number of samples, obtained using a particular thromboplastin versus the World Health Organisation (WHO) international reference preparation for thromboplastin (human combined 67/40). A particular value of ISI, which takes into account the particular method and type of thromboplastin used, is assigned to each PT system, whereby each PT ratio can be translated into a standardised ratio. By employing INR, patients should be able to maintain a satisfactory level of coagulation which is independent of the PT system used. A PT and therefore INR value higher than normal means that the blood is taking longer than usual to form a clot. The normal value for the INR is 1.0 with values recommended between 2.5 and 3.5 for patients with prosthetic heart valves. The value of INR may be used to adjust the warfarin dose to bring a patient to within a recommended range, although other factors such as the levels of Vitamin K may need to be considered.
Another method of measurement of coagulation in either blood or plasma is the Activated Partial Thromboplastin Time Test (APTT). This test is a measure of the time of coagulation that occurs when the intrinsic pathway is activated. This is achieved by the addition of an activator (kaolin) to the sample in the presence of calcium ions and phospholipid (partial thromboplastin). APTT is used to evaluate the intrinsic coagulation pathway which includes the factors I, II, V, VIII, IX, X, XI and XII. Formation of complexes on the surface of the phospholipid enables prothrombin to be converted into thrombin, which results in clot formation.
APTT is used as a routine test for monitoring heparin therapy during surgical procedures, as a preoperative screening test for bleeding tendencies and to assess the overall competence of the patient's coagulation system. This test is commonly carried out in the central laboratory.
The Activated Clotting Time Test (ACT) resembles the APTT test and is used to monitor a patient's coagulation state during procedures that involve the dosing of high amounts of heparin, such as percutaneous transluminal coronary angioplasty (PCTA) and cardiopulmonary bypass surgery. The ACT test is considered as one of the best laboratory tests for the control of heparin therapy, both for patients undergoing treatment for thromboembolic disease and for those on extra-corporeal circulation. For those patients taking heparin, prolongation of the ACT is directly proportional to the concentration of heparin in blood. Monitoring is important and underdosing or overdosing of heparin may result respectively in pathological thrombus formation or serious hemorrhagic conditions.
The Thrombin Time Test (TT) measures the rate of formation of a fibrin clot in plasma by the action of thrombin on fibrinogen, compared to a normal plasma control. The test is performed by adding a standard amount of thrombin to a patient's plasma that has been deprived of platelets and measuring the time for a clot to form. It has been used in the diagnosis of disseminated intravascular coagulation and liver disease and is generally performed in the central laboratory.
Other clotting tests have been developed which target specific factors such as factor VIIIa which is indicative of factor IX deficiency. Another example is an assay for factor VIII, which constitutes a test for haemophilia. Other tests include assays to measure the levels of activation peptide factor IXa, antithrombin, protein C and protein S. Immunochemical assays have also been developed to identify and measure the various markers of coagulation and thrombosis.
Screening for platelet function is an important and common hematological test. Platelets are colourless cell fragments of about 2-4 um in diameter and are present in blood. Normal platelet counts range from 180,000-400,000/uL, however a platelet count of 50,000/uL is sufficient for normal hemostasis. After vascular damage, for example after surgery, higher platelet counts are needed, sometimes in excess of 100,000/uL. The purpose of platelets is to repair gaps in the blood vessel wall by either adhering to themselves or to damaged tissue. When cells become damaged, they release certain chemicals which cause the platelets to change from a discoid to a spherical form and become sticky, known as the aggregation-adhesion reaction.
Platelets are thought to play an important role in the pathogenesis of isechemic heart disease. Acute myocardial infarctions and unstable angina are clinical conditions associated with increased concentrations of certain platelet factors. Furthermore platelet dysfunction is one of the several major causes of bleeding after cardiopulmonary bypass. Platelets are also thought to contribute to the long-term process of atherogenesis by the release of growth factors and platelet function may also be influenced by high and low density lipoproteins. Thus screening for platelet function is an important and common hematological test.
Various instruments have developed for use in the laboratory and as point of care testing (POCT). In addition to this, devices have been developed which allow the patients to home-monitor their blood coagulation. Examples of such are exemplified below.
U.S. Pat. No. 5,534,226 assigned to International Technidyne Corporation, discloses an device and method for performing a coagulation time test on a blood sample whereby the blood is deposited into a capillary via a reservoir disposed within a disposable cuvette. The sample is then caused to reciprocally move within the capillary and blood forced to transverse a restricted region. Coagulation is determined to have occurred when the time required to transverse the restricted region is a predetermined percentage longer than the previous time.
U.S. Pat. No. 6,060,323 assigned to Hemosense, discloses a single use electronic device and test card for the measurement of the coagulation or lysis of a blood sample, typically 15 uL in volume. The sample is caused to contact electrodes, which measure the change in impedance corresponding to the change of viscosity of the sample as it clots.
U.S. Pat. No. 4,849,340, assigned to Cardiovascular Diagnostics, discloses a reaction slide for use with an device for the optical determination of prothrombin time. The reaction slide comprises a reaction chamber containing a dry reagent matrix in which are embedded a plurality of homogeneously distributed magnetic particles. Under the influence of a magnetic field from a permanent magnet, providing a field parallel to the base of the slide the particles are said to lie down against the base of the slide and under the influence of an electromagnet providing a field orthogonal to that of the permanent magnet, the particles are said to stand upright. A resulting change in light intensity due to the light scattering effects of the magnetic particles in their two orientations is detected.
U.S. Pat. No. 5,039,617 assigned to Biotrack discloses a device and method for carrying out the determination of activated partial thromboplastin time (APTT) on a whole blood sample by applying the sample to a capillary track contained in a housing, wherein clotting time is measured by the cessation of blood flow in the capillary track.
U.S. Pat. No. 4,319,194 discloses an aggregometer which is able to carry out platelet analysis on whole blood. Wire shaped electrodes are inserted into the blood sample to which an aggregating agent is added and the change in impedance is recorded as a function of time.